VMA8 encodes a 32-kDa V1 subunit of the Saccharomyces cerevisiae vacuolar H(+)-ATPase required for function and assembly of the enzyme complex

Abstract

The isolated Saccharomyces cerevisiae vacuolar proton-translocating ATPase (V-ATPase) is composed of at least 10 subunits. We have identified VMA8, the gene encoding the 32-kDa subunit of the V-ATPase, by 100% match between the sequences of tryptic peptides and the predicted protein sequence of ORF11. The VMA8 gene contains a 768-base pair open reading frame encoding a 256-amino acid protein with a predicted molecular mass of 29,176 Da. Disruption of VMA8 resulted in a mutant exhibiting pH-sensitive growth, slowed growth under all conditions, and an inability to grow on nonfermentable carbon sources. Vacuolar membranes isolated from vma8 delta yeast cells exhibited no V-ATPase activity. Immunoblot analysis of vma8 delta cells revealed normal levels of both V1 and Vo subunits. Whereas the V1 subunits failed to associated with the vacuolar membrane in vma8 delta cells, the Vo polypeptides were transported to and stable in the vacuolar membrane. Density gradient fractionation revealed that Vma8p associated only with the fully assembled V-ATPase and did not associate with a separate lower density Vo subcomplex fraction. Finally, Vma8p was unable to assemble onto the vascular membranes in the absence of other V1 subunits.